【作者】 陈弟诗； 郭万柱； 徐志文； 陈杨； 李雯； 王小玉；

【机构】 四川农业大学动物生物技术中心；

【摘要】 猪霍乱沙门氏菌C500株不仅可以作为预防猪沙门氏菌病的活疫苗,还可作为运送其他DNA疫苗的优良载体,并通过粘膜免疫诱导产生针对特定抗原的各种免疫应答。为了增强其携带DNA疫苗的免疫效力,本研究在真核表达载体pEGFP-C1的基础上,将其真核启动子CMVIE与原核启动子Ptrc串联,并在其多克隆位点MCS下游引入rrnbT1T2转录终止序列,构建了真、原核双启动子表达载体pEGFPPtrcR。用1×TSS法将其转化C500,得到工程菌C500/pEGFPPtrcR,SDS-PAGE检测了报告基因EGFP的原核表达,该菌在荧光显微镜下能发出强烈绿色荧光,并被证明在体外至少能稳定遗传20代;采用脂质体介导法将质粒pEGFPPtrcR转染真核Vero细胞,EGFP在其胞核和胞浆表达,24h后观察可到明显绿色荧光。结果表明,双启动子表达载体pEGFPPtrcR构建成功,预示其携带的外源基因既可在C500中表达,又可在体细胞中表达,这样能有效提高抗原的表达量从而诱导更强的免疫应答,为研制以C500为载体的新型DNA疫苗的发展开辟了一个新的途径。更多还原

【Abstract】 As an attenuated vaccine preventing piglet from paratyphoid,Salmonella choleraesuis C500 strain can also be used as a live vector of other DNA vaccines,through mucosa immunization,immune response to specific antigens carried by it can be induced.To enhance the immune efficiency of DNA vaccine it carried,Prokaryotic promoter Ptrc was inserted into the eukaryotic promoter CMVIE down stream of eukaryotic expression plasmid pEGFP-C1,and transcription terminator rrnbT1T2 was inserted into the multiple clone site’(MCS’s) downstream of pEGFP-C1,then the dual-promoter expression vector pEGFPPtrcR was constructed. Using 1×TSS method,the recombinant plasmid was transformed into C500,C500/pEGFPPtrcR was obtained.SDS-PAGE was used to detect the expression of report gene EGFP;strong green fluorescence can be observed under fluorescent microscope.The stable passages of this recombinant bacteria were at least 20 generations in vitro.Mediated by liposome,the plasmid pEGFPPtrcR was transfected into Veto cell,and after 24h,green fluorescent was observed,showing the expression of EGFP in nuclei and endochylema. The result manifested that the construction of dual-promoter expression vector pEGFPPtrcR was successful and indicated that the heterologous gene carried by it was able to express in salmonella strain C500 and also in somatocytes,which can increase the antigen expression amount.The increased expression amount of antigen can induce stronger immune response.This research provided a foundation for the research of new DNA vaccine using salmonella C500 as carrier.更多还原